S284: HEPATOCYTE TOLL-LIKE RECEPTORS MEDIATE THE HEPCIDIN INFLAMMATORY RESPONSE TO PATHOGENS AND PATHOGEN-DERIVED LIGANDS
Authors: Katharina Bonitz, Silvia Colucci, Richard Sparla, Ruiyue Qiu, Sandro Altamura, Katja Muedder, Stefan Zimmermann, Martina U. Muckenthaler, Oriana Marques
Journal: HemaSphere (2023)

Background: Iron restriction is an innate immune mechanism that limits the replication of extracellular pathogens. During an infection, innate immune cells detect pathogen-associated molecular patterns (PAMPs) via Toll-like receptors (TLRs) and produce proinflammatory cytokines that contribute to systemic inflammation. Macrophage-derived Interleukin (IL)-6 is detected by hepatocytes, promoting the acute-phase response and the production of the master regulator of systemic iron flows – hepcidin. Hepcidin blocks iron export via ferroportin from duodenal enterocytes and macrophages, a process that leads to iron retention, reduced plasma iron availability and anemia of inflammation (AI). Due to the production of proteins participating in pathogen removal – such as hepcidin – hepatocytes are increasingly recognized as a critical cell type of the innate immune system. Aims: We asked the question whether TLRs expressed on hepatocytes recognize PAMPs, activate hepcidin expression and contribute to iron restriction. Methods: Primary murine hepatocytes were stimulated with ligands targeting TLRs 1-9 and mRNA expression and cytokine secretion was analyzed. Signaling pathways were inhibited by treatment with pharmacological inhibitors or RNA interference of upstream mediators and confirmed by protein analysis of phosphorylation targets. A hepatocyte:macrophage co-culture system was established with primary murine hepatocytes and bone-marrow derived macrophages. Co-culturing conditions included both wild-type (WT) and Tlr2/6 knock-out (KO) hepatocytes and/or macrophages to allow for the dissection of the relative contribution of each cell type to the downstream inflammatory response. Ex vivo liver perfusion was performed with 1 µg of FSL1 or 108 particles of S. aureus followed by standard preparation of primary murine hepatocytes. Results: Stimulation of murine hepatocytes with most TLR ligands increased cytokine expression. By contrast, increased hepcidin expression was limited to the stimulation with the TLR5 ligand flagellin and the TLR2/6 ligand FSL1. Increased hepcidin expression in hepatocytes in response to FSL1, heat-killed S. aureus (HKSA) and B. abortus (HKBA) started at an early time-point and was dependent on both TLR2 and partially, TLR6, but was independent of autocrine regulation by IL-6 and IL-1β. Interestingly, FSL1, flagellin, HKSA and HKBA-mediated hepcidin upregulation was attenuated by inhibition of the c-Jun N-terminal Kinase (JNK) and Extracellular signal-Regulated Kinase (ERK) pathways, linking novel pathways to the inflammatory hepcidin response. The relevance of TLR2 and 6 was further substantiated in a hepatocyte:macrophage co-culturing system, where we demonstrate that FSL1-mediated hepcidin upregulation is independent of macrophage cytokine secretion at an early time-point, but rather dependent on the presence of TLR2/6 on hepatocytes. Finally, we asked if PAMPs or pathogens could diffuse through the hepatic sinusoids and be recognized directly by hepatocytes. Ex vivo liver perfusion of WT and Tlr2/6KO mice with FSL1 and HKSA revealed that PAMPs and pathogens can pass the sinusoidal barrier and reach hepatocytes to cause hepcidin upregulation in a TLR2/6-dependent manner. Summary/Conclusion: In conclusion, our data show that hepatocytes can directly recognize PAMPs and pathogens and in this way promote hepcidin upregulation in a macrophage and cytokine independent manner. This response is expected to contribute to iron restriction and the development of AI. The recognition of hepatocytes as innate immune cells that detect exogenous and endogenous PAMPs, places hepatocytes in the spotlight as direct drivers of iron restriction in specific infectious settings. Keywords: Hepcidin, Anemia, Innate Immunity, Inflammation